It is well known that the production of peptides of less than about 100 amino acids in length by expression of peptide-encoding DNA in a recombinant host cell such as E. coli is plagued by enzymatic degradation of the expressed peptide within the host cell, which often results in partial or complete loss of the peptide. The most commonly employed means to overcome this problem is to insolubilize the peptide within the host cell. This can be affected by expressing the peptide as a chimeric protein in which the peptide is linked to a fusion partner. Normally, the fusion partner will be fused to the N-terminus of the peptide. The chimeric protein forms inclusion bodies within the cell, within which the peptide is protected from degradation by proteolytic enzymes. Once the inclusion bodies are recovered from the host cell, the peptide must be separated from the leader sequence, purified and recovered in an active form. Cleavage from the leader sequence may be accomplished by placing a sequence of amino acids at the junction of the leader and the peptide which are specifically recognized and cleaved under appropriate conditions, e.g. acid cleavage or enzymatic cleavage.
For example, introduction of acid-labile aspartyl-proline linkage between the two segments of a chimeric protein facilitates cleavage at low pH. This technique does not work if the product peptide, which is to be cleaved from the polypeptide, is not acid-labile. Chimeric proteins comprising hormones such as insulin and somatostatin have been cleaved with cyanogen bromide, which is specific for the carboxyl side of methionine residues. This method is not suitable when the product peptide contains methionine residues.
Cleavage of chimeric proteins by site-specific proteolysis has also been investigated. Chimeric proteins containing a chicken pro alpha.-2 collagen linker could be specifically degraded by purified microbial collagenase to release the components of the chimeric protein. Use of proteolytic enzymes is expensive, product peptide cleavage yield is frequently low, and it can prove difficult to separate the enzyme from a desired peptide product. Other methods for purification and recovery of a desired recombinant protein include construction of a poly-arginine tail at the C-terminus of the polypeptide. The arginine residues increase the overall basicity of the protein, which facilitates purification by ion exchange chromatography. Subsequent removal of the poly-arginine tail by carboxypeptidase B regenerates the desired protein and allows purification from basic contaminants due to the reduction in pI of the desired protein.
Acid cleavage can be accomplished by placing a specific dipeptide at the junction of the leader sequence and the peptide. Selection of the second amino acid will determine the rate at which the dipeptide bond is cleaved under acidic conditions. Of course, if the desired peptide contains any internal dipeptide sequences that are acid cleavable, then the cleavage site at the junction of the leader and the peptide must undergo acid cleavage at a substantially greater rate than the internal cleavage in order to avoid unacceptable loss of yield.
Zhu et al., J. Am. Chem. Soc. 116: 5218 (1994), describe selective cleavage of cytochrome-c at S-hemo-Cysteinyl-Histidine (Cys(hemo)-His) using certain palladate(II)(Pd II) complexes under acidic conditions. Under the reaction conditions described in Zhu et al., cleavage of cytochrome c took 2 days at 40° C. and resulted in a cleavage yield of only 35-50%. (SDS-PAGE analysis in fact indicated some degree of non-specific cleavage; as the molar ratio of Pd to protein was increased from 1:1 to 4:1, there was an indication of cleavage at a site other than the Cys(hemo)-His sequence.)
Zhu et al. state at p. 5220 that 100 mM HBF4, HClO4, and CF3COOH, or 70% formic acid, cleaved cytochrome-c. The reference thereafter concludes that cleavage was inhibited by the presence of chloride ions, a notable drawback as proteins purified from biological systems will almost invariably contain chloride ions. Notably, Zhu et al. at p. 5219 conclude that the rate of hydrolysis depended on conformational aspects of cytochrome c (i.e., cleavage was thought to be affected by the size of the cleaved peptide fragment and hence, the sequence of the polypeptide to be cleaved).
Dou, et al., “Preliminary Study On The Cleavage Of Chimeric protein GST-CMN With Palladate(II) complex”, Prep. Biochem & Biotechnol., 301(1): 69-78 (2000) (“Dou, et al.”), describe palladate promoted hydrolytic cleavage of a cecropin CMIV chimeric protein using formic acid, acetic acid, phosphoric acid and HBF4 and [Pd(en)(H2O)2]2+Dou, et al. sought to cleave their chimeric protein specifically at two cleavage sites: Cys-His-Lys and Cys-His-Arg. According to Dou, et al., p. 76, only the HBF4 reaction media cleaved the chimeric protein at the experimental conditions of 40° C. and reaction time of 48 hours. The cleavage in HBF4 was said to be pH-dependent or independent depending on the amino acid adjacent to Cys-His: cleavage at Cys-His-Lys was pH-independent while cleavage at Cys-His-Arg was pH-dependent. Id. Further, cleavage at either site in HBF4 was temperature dependent; when the temperature was increased to 60° C. the chimeric protein solubilized and it was no longer possible to cleave selectively. Id. Dou, et al. understood that their reaction would be strongly inhibited by the presence of chloride ions and employed an extra ion-exchange chromatography purification step before the cleavage reaction.
Read in context, Dou et al. and Zhu et al. would suggest that palladate promoted hydrolytic cleavage of polypeptides is not assured in concentrated acidic organic media, but instead is dependent upon the sequence of the polypeptide to be cleaved, reaction temperature and the possible inhibitory effects of chloride-containing species, including highly useful chloride-containing palladates. Dou et al. imply that solubilization of their chimeric protein results in a loss of cleavage specificity.
In sum, known hydrolytic polypeptide cleavage processes would suggest that even in concentrated acidic media, either the sequence of the polypeptide, the reaction media temperature or ionic species present in the reaction media could limit cleavage yield and specificity.